Direct identification of rat iNKT cells reveals remarkable similarities to human iNKT cells and a profound deficiency in LEW rats

Abstract

iNKT cells are a particular lymphocyte population with potent immunomodulatory capacity; by promoting or suppressing immune responses against infections, tumors, and autoimmunity, iNKT cells are a promising target for immunotherapy. The hallmark of iNKT cells is the expression of a semiinvariant TCR (with an invariant α-chain comprising AV14 and AJ18 gene segments), which recognizes glycolipids presented by CD1d. Here, we identified iNKT cells for the first time in the rat using rat CD1d-dimers and PLZF staining. Importantly, in terms of frequencies (1.05% ± 0.52 SD of all intrahepatic αβ T cells), coreceptor expression and in vitro expansion features, iNKT cells from F344 inbred rats more closely resemble human iNKT cells than their mouse counterparts. In contrast, in LEW inbred rats, which are often used as models for organ-specific autoimmune diseases, iNKT cell numbers are near or below the detection limit. Interestingly, the usage of members of the rat AV14 gene family differed between F344 and LEW inbred rats. In conclusion, the similarities between F344 rat and human iNKT cells and the nearly absent iNKT cells in LEW rats make the rat a promising animal model for the study of iNKT cell-based therapies and of iNKT-cell biology.

The hypervariable region 4 (HV4) and position 93 of the α chain modulate CD1d‐glycolipid binding of iNKT TCRs

“…While the rAV14S6 93A containing iNKT TCR revealed substantial binding to rCD1d, there was nearly no binding to mCD1d or huCD1d detectable (Fig. 1C), which is in accordance with previous reports describing the lack of species cross-reactivity of rat iNKT cells [15,22]. Intriguingly, the rAV14S2 93G-containing iNKT TCR exhibited a lower rCD1d but an increased mCD1d and huCD1d binding when compared to rAV14S6 93A (Fig.…”

“…Analysis of AV14/AJ18 rearrangements from rat revealed the existence of noncanonical iNKT TCRα sequences containing glycine instead of the germline encoded alanine at position 93 [20,22]. Interestingly, glycine is the amino acid present in the canonical mouse iNKT α chain sequence [5,6].…”

“…Since we showed that the HV4α and not the CDR2α was mainly responsible for differences in rat CD1d binding, the original grouping of those segments into type 1 and 2, depending on their CDR2 region, appears obsolete [20]. Instead a functional grouping should consider the HV4 region, as the differences within this region are spread across all rat AV14 gene segments [22].In analogy, the substitution of lysine 68 to an alanine seen for the mouse iNKT TCRα can be explained on the basis of the current structure data. Here, lysine 68 is engaged in an h-bond network, which likely stabilizes the loop conformation of CDR1α of the mouse TCR.…”

“…In addition, variability within the TCRα chain of primary iNKT-cell populations has been observed at position 93 (V-J transition), i.e. within a substantial proportion of TCRα chain sequences the germline encoded alanine had been replaced by glycine [20,22]. Together, this highlights the possibility of increased variability within a classically invariant system.…”

“…Rats have a CD1d gene whose expression pattern on hematopoietic cells is nearly identical to that of mouse CD1d, different BV8S2 homologues (BV8S2 and BV8S4), and generate typical AV14/AJ18 rearrangements [15,[18][19][20][21]. A direct identification of rat iNKT cells has been possible only recently by staining of primary rat iNKT cells with rat CD1d dimers [22]. We found that iNKT cells from the F344 rat strain show a striking similarity to human iNKT cells with respect to low frequency, expression of CD8α, and robust ex vivo expansion, encouraging the use of rats as a model for iNKT cell based investigations.…”